Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.

2',7'-Dichloro- and 2',7'-difluorofluoresceins are superior alternatives to underivatized fluorescein. Although several studies characterizing their condensed-phase photophysical properties have been reported, little is known about their intrinsic characteristics. Here, the gas-phase properties of three charge states of each fluorescein are characterized using a quadrupole ion trap mass spectrometer which has been modified for spectroscopy. Electronic action spectra, constructed by monitoring the extent of photodissociation as a function of excitation wavelength, indicate that the gaseous dianions and cations resemble their solution-phase counterparts. In contrast, a large shift in the electronic action spectra of the monoanions indicates the presence of a different tautomer in the gas phase than that present in solution. The gaseous monoanion is deprotonated on the xanthene ring, rather than being deprotonated on the pendant group as found in soluion. The dianions and cations do not emit detectable fluorescence in the gas phase. In contrast, the monoanions do fluoresce, but the emission intensity is low and the spectra are broad. This work illustrates the effect of halogenation on the intrinsic properties of the dyes and provides useful fundamental understanding that promises to aid the development more robust fluorescent dyes.

Fluorescein is used extensively for visualization and diagnostics in biological and medical applications. The popularity of fluorescein, which has been studied for over a century, [1] arises from its bright fluorescence and its ease of conjugation to biomolecules.[2] Fluorescein exists in up to seven different pHdependent states: [3a] three neutral forms and four charged forms (Scheme 1). These forms each have different excitation and fluorescence emission properties, some of which are strongly solvent-dependent. To better understand the effect of the microenvironment on the spectroscopic properties of fluorescein, knowledge of its intrinsic (solvent-free) properties is crucial. Herein, we use the isolation capabilities of trapping mass spectrometry to individually probe the spectroscopy of the three fluorescein charge states. An unexpected result is that the brightest form of fluorescein in solution, the dianion, does not fluoresce significantly in the gas phase.The absorbance and the quantum yield of fluorescein in solution vary significantly with the protonation state. The fluorescein dianion ([FlÀ2 H] 2À ; Scheme 1) has the highest molar absorptivity (ca. 10 5 m À1 cm À1 at l ab max ¼ 490 nm in water) and fluorescence quantum yield (0.92).[3] The monoanion ([FlÀH] À ) is also fluorescent, but has a lower absorptivity (two maxima of ca. 30 000 m À1 cm À1 at l ab max ¼ 450 and 470 nm in water) and fluorescence quantum yield (0.37).[3] Fluorescence upon excitation of cationic (and neutral) fluorescein is observed; however, this fluorescence is believed to occur through deprotonation in the excited state, thus forming the fluorescent excited monoanionic species. The effective fluorescence quantum yield for the fluorescein cation is 0.18, which reflects both the efficiency of the excited state proton transfer reactions and the quantum yield of the monoanion.[3a]The fluorescein dianion exhibits significant solvatochromism.[4] This observation was first reported by Martin, [4a] who showed that as the solvent was changed from H 2 O to dimethyl sulfoxide (DMSO), the absorption maximum for the dianion shifted from 490 nm to 520 nm. The observed solvatochromism was attributed to the hydrogen bonds between the fluorescein dianion and the solvent being stronger in the ground state than in the excited state, thus increasing the gap between the S 0 and S 1 electronic energy levels as the hydrogen-bonding ability of the solvent increases.Whereas there is a breadth of information on the behavior of fluorescein in solution, studies of the properties of fluorescein in the gas phase have been limited to computational work.[5] Jang et al.[5b] have performed electronic structure theory calculations for nine different fluorescein tautomers in vacuo and in DMSO and water. Computations at the B3LYP/6-31 + + G** level of theory with a Poisson-Boltzmann continuous solvation approach showed that the most stable conformers of cationic and dianionic fluorescein in solution are similar to the most stable gas-phase forms. However, dep...

Fluorescein (FL) and its derivative 2',7'-dichlorofluoroescein (DCF) are well-known fluorescent dyes used in many biological and biochemical applications. Although extensive studies have been carried out to investigate their chemical and photophysical properties in different solvent media, little is known about their intrinsic behaviors in the gas phase. Here, infrared multiple photon dissociation (IRMPD) action spectra are reported for the three charged prototropic forms of FL and DCF and compared with computed IR spectra from electronic structure calculations. In each case, the measured spectra show good agreement with the calculated spectra of the lowest energy computed conformer. Moreover, the major bands of the monoanion IRMPD spectra show striking similarities to those of the dianions and are quite different from those of the cations. These experimental results clearly indicate that the gaseous monoanions are predominantly deprotonated on the xanthene chromophore, rather than the benzoate deprotonation site favored in solution. Investigations such as this, which provide a better understanding of intrinsic properties of ionic dyes, forms a baseline from which to elucidate solvent effects and will aid the rational design of dyes possessing desirable fluorescence properties.

Fluorescein is used extensively for visualization and diagnostics in biological and medical applications. The popularity of fluorescein, which has been studied for over a century, [1] arises from its bright fluorescence and its ease of conjugation to biomolecules. [2] Fluorescein exists in up to seven different pHdependent states: [3a] three neutral forms and four charged forms (Scheme 1). These forms each have different excitation and fluorescence emission properties, some of which are strongly solvent-dependent. To better understand the effect of the microenvironment on the spectroscopic properties of fluorescein, knowledge of its intrinsic (solvent-free) properties is crucial. Herein, we use the isolation capabilities of trapping mass spectrometry to individually probe the spectroscopy of the three fluorescein charge states. An unexpected result is that the brightest form of fluorescein in solution, the dianion, does not fluoresce significantly in the gas phase.The absorbance and the quantum yield of fluorescein in solution vary significantly with the protonation state. The fluorescein dianion ([FlÀ2 H] 2À ; Scheme 1) has the highest molar absorptivity (ca. 10 5 m À1 cm À1 at l ab max ¼ 490 nm in water) and fluorescence quantum yield (0.92). [3] The monoanion ([FlÀH] À ) is also fluorescent, but has a lower absorptivity (two maxima of ca. 30 000 m À1 cm À1 at l ab max ¼ 450 and 470 nm in water) and fluorescence quantum yield (0.37). [3] Fluorescence upon excitation of cationic (and neutral) fluorescein is observed; however, this fluorescence is believed to occur through deprotonation in the excited state, thus forming the fluorescent excited monoanionic species. The effective fluorescence quantum yield for the fluorescein cation is 0.18, which reflects both the efficiency of the excited state proton transfer reactions and the quantum yield of the monoanion. [3a] The fluorescein dianion exhibits significant solvatochromism. [4] This observation was first reported by Martin, [4a] who showed that as the solvent was changed from H 2 O to dimethyl sulfoxide (DMSO), the absorption maximum for the dianion shifted from 490 nm to 520 nm. The observed solvatochromism was attributed to the hydrogen bonds between the fluorescein dianion and the solvent being stronger in the ground state than in the excited state, thus increasing the gap between the S 0 and S 1 electronic energy levels as the hydrogen-bonding ability of the solvent increases.Whereas there is a breadth of information on the behavior of fluorescein in solution, studies of the properties of fluorescein in the gas phase have been limited to computational work. [5] Jang et al. [5b] have performed electronic structure theory calculations for nine different fluorescein tautomers in vacuo and in DMSO and water. Computations at the B3LYP/6-31 + + G** level of theory with a Poisson-Boltzmann continuous solvation approach showed that the most stable conformers of cationic and dianionic fluorescein in solution are similar to the most stable gas-phase forms. Howe...